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Volume 271, Number 43, Issue of October 25, 1996 pp. 26794-26802
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Myristoylation Does Not Modulate the Properties of MARCKS-related Protein (MRP) in Solution

(Received for publication, June 25, 1996, and in revised form, August 5, 1996)

Enrico Schleiff , Arndt Schmitz , R. A. Jeffrey McIlhinney ^¶ , Stéphane Manenti and Guy Vergères

From the  Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland, the ^¶ Medical Research Council, Anatomical Neuropharmacology Unit, Department of Pharmacology, University of Oxford, South Parks Road, Oxford OX1 3TH, United Kingdom, and  INSERM CJF 95-10, CHU Purpan, 31059 Toulouse, France

The members of the yristoylated lanine-ich inase ubstrate (MARCKS) family are proteins essential for brain development and phagocytosis. MARCKS proteins bind to actin filaments and calmodulin (CaM) and are phosphorylated by protein kinase C. In order to investigate how these interactions are regulated, we have characterized the properties of both the myristoylated (myr) and unmyristoylated (unmyr) forms of recombinant MARCKS-related protein (MRP), a 20-kDa member of the MARCKS family.

Ultracentrifugation and circular dichroic spectroscopy reveal that MRP is an elongated protein, with an axis ratio estimated between 7 and 12 and with an apparent random coil conformation. MRP binds to CaM with high affinity (K_d,myr = 4 nM; K_d,unmyr = 7 nM) and with a second order rate constant, k_+1,unmyr, of 1.6 × 10⁸ M¹ s¹. In contrast to classical ligands such as the myosin light chain kinase, binding of MRP to CaM does not induce the formation of an -helix in MRP. The catalytic subunit of protein kinase C (PKM) phosphorylates myr MRP with high affinity ([S]_0.5 = 3.5 µM), positive cooperativity (n_H = 2.5) and a turnover number of 130 min¹. CaM inhibits the phosphorylation of myr MRP with a half-maximum rate of phosphorylation at a [CaM]/[MRP] ratio of 0.7, indicating that CaM might efficiently regulate the phosphorylation of MRP in vivo. Interestingly, Ca²⁺ inhibits the binding of MRP to CaM as well as its phosphorylation by PKM in the millimolar concentration range, suggesting that MRP has a weak affinity for Ca²⁺.

Finally, unmyr MRP can be stoichiometrically myristoylated by N-myristoyl transferase in vitro. Since neither binding of CaM nor phosphorylation by PKM inhibits myristoylation, the N terminus of unmyr MRP is exposed on the surface of the protein and is well separated from the effector domain. In view of the observations that unmyr and myr MRP do not exhibit significant differences in their properties in solution, the function of myristoylation is most probably to modulate the interactions of MRP with membranes.

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